Filoviruses are filamentous enveloped viruses that cause hemorrhagic fever with mortality rates approaching 90%. Hence there is a pressing need to develop therapeutics to combat natural, accidental or intentional infections by these deadly viruses. Since virus entry is an ideal target for therapeutic intervention, the long-term objective of this work is to develop agents to block filovirus entry. Our focus is on the membrane fusion step of filovirus entry, which is mediated exclusively by the viral glycoprotein (GP). Intriguingly the way in which GP is triggered does not conform to any known viral fusion triggering process. Several additional observations bear on the novel means by which filovirus fusion is triggered. The first is our recent discovery that mild reducing conditions, which prevail at the site of filovral fusion in endolysosomes (LE/Lys), are sufficient to trigger the primed Ebola virus GP to bind to target membranes and induce fusion. The second is that cystinosin, a cystine transporter, maintains the reducing environment in LE/Lys. The third is that although it is essential for filovirus entry and binds to primed GP in LE/Lys, we have found that Niemann-Pick C1 (NPC1) is not sufficient to trigger GP for fusion at low pH. The fourth is that LE/Lys from NPC1 null mice were reported to have more cystine (i.e., be less reducing) than those from WT mice. These observations have led us to an exciting new working hypothesis for filovirus fusion: Upon entry into LE/Lys, cathepsins prime GP by removing its heavily glycosylated domains, thereby exposing a binding site for NPC1. After binding to NPC1, GP is triggered by LE/Lys reducing potential, which is maintained by cystinosin. NPC1 plays multiple roles: it protects primed GP from inactivating proteolysis; it targets fusion to the proper (outer) LE/Lys membrane, and it helps maintain adequate reducing potential in LE/Lys. We will test our new model by addressing three Specific Aims: (1) Determine the contributions of NPC1 and disulfide reducing potential to filovirus fusion triggering; (2) Determine roles for NPC1 upstream of fusion triggering; and (3) Determine if cystinosin is required for filovirus fusion. Our experimental design will employ a novel preparation of 'multi-purpose' filoviral like particles (VLPs), which can monitor, with robus quantitative flow cytometric-based assays, major steps of virus entry: particle binding, internalization, fusion, and access to the cytoplasm. VLPs will be used in conjunction with WT and NPC1 and cystinosin deficient cells to test each facet of our model. Our work will have high basic science impact by (a) unveiling a novel viral fusion mechanism that may be used by other pathogenic viruses and by (b) exploring a new biological role for acid-dependent endosomal reduction, an underappreciated process that likely has broader roles in endocytosis. Moreover, our work will have high translational impact. Filoviruses are classed as category A priority pathogens because of their high human case mortality rates, ease of person-to- person spread, and lack of confirmed therapeutics. Our studies will advance NPC1, and perhaps cystinosin, as new host cell targets for the development of therapeutics to combat filovirus infections.